Interpretive Summary: The aim of this work is to gain insight into how cellular metabolism adjust to the over-expression of a seed storage protein, HMW-glutenin subunit Dx5, in developing transgenic wheat grains. We analysed the data using several statistical methods to compare the transformed transgenic lines over-expressing the storage protein from those containing the vector alone.

Technical Abstract:
The primary aim of this work was to evaluate potential changes in the metabolic network of transgenic wheat grain due to over-expression of the gene encoding the high-molecular-weight (HMW) glutenin Dx5-subunit. We used GC-MS and multivariate analyses to compare the metabolite profiles of developing caryopses of two independent transformants over-expressing Dx5 and two independent transformants expressing only the selectable-marker gene (controls). Principal component analysis (PCA) of the data showed no distinction between the Dx5 transformants and the controls suggesting that their metabolite compositions were similar. The levels of only 3 metabolites, guanine, 4-hydroxycinnamic acid and Unknown 071306a, were altered due to Dx5 expression after correction for false discovery rates (p< 0.0005). Discriminant function analysis (DFA) and correlative analyses of the metabolites for each genotype showed that Dx5-J, which had the highest level of Dx5 protein in ripe caryopses, could be distinguished from the other genotypes. Furthermore, half of the metabolites with altered correlative patterns in the Dx5 transformants are compounds metabolically linked to glutamate and glutamine. These amino acids constitute 45% of the dry weight of the Dx5 protein and an adjustment by linked metabolites may be part of the strategy to compensate for over-expression of Dx5. Cross-comparison of the transformed controls to each other, and to the Dx5 genotypes allowed us to determine that approximately 50% of the metabolic changes in the Dx5 genotypes were potentially due to variations arising from gene transformation.